This mentored research career development proposal (K99/R00) is designed to provide the candidate advanced training, expert mentorship, and hands-on research experience to facilitate a pathway to an independent research career. The candidate completed graduate training in multimodal functional and spectroscopic neuroimaging and investigated the neurobiological consequences of nicotine and opioid abuse. On this solid foundation of clinical neuroscience training, the candidate proposes to develop expertise in positron emission tomography (PET) imaging to investigate the molecular underpinnings of opioid use disorder (OUD). To achieve this goal, the candidate will complete a comprehensive two-year plan which will provide rigorous training in three key areas: 1) PET imaging methodology and analysis; 2) clinical research among opioid dependent individuals; and 3) responsible conduct in research. The mentorship team has expertise in PET imaging (Drs. Richard Carson and Kelly Cosgrove) and opioid use disorder (Dr. David Fiellin) needed to facilitate an exemplary training experience. This plan will be completed in a highly-productive and supportive training environment at the Yale University School of Medicine. This K99/R00 Pathway to Independence application will enable the candidate to develop into an independent investigator using multimodal neuroimaging techniques to study substance use disorders, especially OUD. The societal consequences of the current OUD epidemic are enormous. There is a tremendous need to investigate novel treatment targets. Recently, there has been increased interest in the neuroimmune system. Opioids are known to exert acute proinflammatory effects in both the periphery and brain. Opioid-induced neuroinflammatory responses may play an important role in the development and perpetuation of OUD. Preclinical and clinical research has linked opioid-induced neuroimmune responses to opioid craving, opioid- seeking behavior, morphine tolerance, and opioid withdrawal symptoms. In this application, we propose to use PET imaging to investigate opioid effects on the in vivo neuroimmune system in two separate and non- contingent studies. [11C]PBR28 PET imaging of 18kDa translocator protein (TSPO) levels is widely-used to study the in vivo neuroimmune system. In Aim 1, we will use [11C]PBR28 PET imaging to quantify acute opioid- induced neuroimmune responses among healthy volunteers using a novel human laboratory paradigm. In Aim 2, we combine PET TSPO imaging with proton magnetic resonance spectroscopy quantification of myo-Inositol levels, a putative glial marker. Together, this multimodal neuroimaging approach will be used to evaluate the in vivo neuroimmune system of early-treatment OUD patients vs. well-matched controls. These studies will clarify the neuroimmune consequences of acute opioid administration and chronic opioid abuse. Elucidation of the role of the neuroimmune system in OUD may motivate novel treatment strategies and improve relapse rates.